Comprehensive Virome Profiling of Apple Mosaic Disease-Affected Trees in Iran Using RT-PCR and Next-Generation Sequencing

Abstract

Apples (Malus domestica), one of Iran's oldest cultivated fruit crops, hold considerable economic importance. In this study, 170 apple leaf samples representing various commercial cultivars were collected across the country. RT-PCR screening targeted five common apple-infecting viruses and two viroids: apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple green crinkle-associated virus (AGCaV), apple mosaic virus (ApMV), apple scar skin viroid (ASSVd), and hop stunt viroid (HSVd). To identify additional or novel agents, 40 RT-PCR-negative samples were pooled into two composite groups and analyzed using next-generation sequencing (NGS). NGS was also performed on individual samples with mixed infections to retrieve full genomes. RT-PCR confirmed the presence of ACLSV, ASPV, ASGV, AGCaV, ApMV, and HSVd. NGS further revealed three additional pathogens: citrus concave gum-associated virus (CCGaV), apple hammerhead viroid (AHVd), and apricot vein clearing-associated virus (AVCaV), which were subsequently detected across the collection by RT-PCR. AGCaV was most prevalent (47.6%), followed by ACLSV (45.8%), HSVd (27.6%), AVCaV (20.5%), ASGV (17%), AHVd (15.2%), ASPV (14.1%), CCGaV (4.7%), and ApMV (3.5%). Mixed infections occurred in 67% of samples. Phylogenetic analysis based on CP genes (ACLSV, ASGV, AGCaV) and full genomes (AVCaV, AHVd) clustered Iranian isolates together, suggesting a common origin. This is the first report in Iran of AGCaV, CCGaV, ApMV, and AVCaV in apple, and notably, the first global report of AVCaV in a non-Prunus host. The findings provide the first comprehensive assessment of the sanitary status of apple trees in Iran.

Keywords: detection; molecular characterization; phylogenetic analysis; viroids; viruses.

Figure 1

Map showing the locations of sampling sites located in four provinces of Iran. The samples were collected from the Central and Northern regions in Iran at the 20 locations indicated by dots on the map.

Figure 2

Apple leaves showing symptoms of interveinal chlorosis (a); vein banding (b); mosaic (c); deformation with necrotic ring spot on cultivar Golden Delicious (d); mosaic with necrotic leaf spots on cultivar Red Delicious (e); mosaic, deformation and yellowing on samples subjected to NGS (f).

Figure 3

Phylogenetic tree based on the complete CP gene nucleotide sequences of 3 Iranian isolates: (a) ACLSV, (b) ASGV, and (c) AGCaV, together with homologous sequences retrieved from the NCBI database. Isolates are labeled with accession number/isolate name/country. Trees were constructed using MEGA 11.0 with the maximum likelihood (ML) method and the Kimura two-parameter model, with 1000 bootstrap replicates. Bootstrap values >70% are shown. Scale bars represent the number of nucleotide substitutions per site. Iranian isolates detected in this research are marked with black dots. Outgroup sequences used were Asian prunus virus 1 (KX962059) for ACLSV, Rubber tree capillovirus 1 (NC_076715) for ASGV, and Grapevine foveavirus A (NC_077040) for AGCaV. See Table 1 and Table 4 for accession details.

Figure 4

Phylogenetic trees based on the full-length genomic sequences of (a) AHVd isolates from apple trees in Mazandaran and Tehran provinces, and (b) AVCaV isolates from apple trees in Tehran province, Iran, together with homologous sequences retrieved from the NCBI database. Isolates are labeled by accession number/isolate name/country. Trees were constructed in MEGA 11.0 using the maximum likelihood (ML) method with the Kimura two-parameter model and 1000 bootstrap replicates. Only bootstrap values above 70% are shown. Scale bars represent the number of nucleotide substitutions per site. Iranian isolates detected in this research are marked with black dots. Hop stunt viroid (HSVd; ON058261) and Cherry virus A (KY445749) were used as outgroup sequences for the AHVd and AVCaV phylogenies, respectively. See Table 1 and Table 4 for accession numbers.